Method and device for air disinfection and sterilization

ABSTRACT

A method for decontaminating bioaerosol with high concentrations of bacterial, viral, spore and other airborne microorganisms or biologic contaminants, in flight at high flow rates. A plasma screen created across the flow of air contaminated with airborne biologic agents renders contaminants non-culturable within millisecond. The technology may cooperate with heating, ventilation, and air conditioning (HVAC) systems. It may be particularly beneficial in preventing bioterrorism and the spread of toxic or infectious agents, containing airborne pandemic threats such as avian flu, sterilizing spaces such as hospitals, pharmaceutical plants and manufacturing facilities, treating exhaust ventilation streams, minimizing biological environmental pollutants in industrial settings, improving general air quality, and preventing sick building syndrome.

STATEMENT OF GOVERNMENT INTEREST

This invention was reduced to practice with Government support a U.S.Army Medical Research Acquisition Activity; Cooperative Agreement W81XWH04-1-0419. The Government is therefore entitled to certain rights tothis invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to a method and device for decontaminatinghigh concentrations of bacterial bioaerosols, viral bioaerosols andother airborne microorganisms in flight at high flow rates. Theinvention is particularly applicable to the Heating, Ventilation and AirConditioning (HVAC) industry and bioterrorism defense industry.

2. Description of the Related Technology

The escalating threat of airborne biologic and bioterrorism agents,present a need for robust technologies and methods to mitigate thespread of airborne contaminants. Events such as the avian flu pandemic,the 1976 Legionnaires outbreak in Philadelphia and the 2001 anthraxterrorism in the United States demonstrate the ability to rapidly spreadbiologic contaminants through ventilation systems.

To address these concerns, scientists are focusing on non-thermalplasma-based technologies, which have previously proven successful indeactivating microorganisms, such as viruses and bacteria, in solutionand on surfaces. Plasma has proven to be useful as a microbialdisinfectant in many surface sterilization studies and it can bedelivered with low power consumption, as a non-thermal discharge that isrelatively easy to construct requiring simple power supplies.

Decontamination of microorganisms in flight using non-thermal plasmatechnology, however, has not been effectively implemented. Plasma-basedair decontamination has only been found effective when coupled with highefficiency particulate air (HEPA) filters, which trap and killmicroorganisms. HEPA filters, however, are inefficient at trappingsubmicron-sized airborne microorganisms. Moreover, HEPA filters alsocause significant pressure losses in heating, ventilation, and airconditioning (HVAC) systems, generating high energy and maintenancecosts. The filters function as a surface on which contaminants arecaptured; therefore, the prior art methodologies are, in essence, thesame as standard plasma surface sterilization. Numerous technologies,such as those disclosed in Chinese Patent no. 02655913Y, U.S. Patentapplication publication no. 2004/0037736A1 and International patentapplication publication nos. WO 03/063914 A2, WO 05/067984 and WO06/003382 A1, similarly sterilize air by directing plasma emissions at afilter surface, which entraps the biologic contaminants.

Apart from treatments in solution or on surface, there remains a need todevelop a means for in flight plasma-based decontamination so as to beable to deactivate microorganisms in the air while in motion. This maybe particularly useful for sterilizing ventilation systems andpreventing the spread of airborne biologic agents.

In Michael J. Gallagher, et. al., “Non-Thermal Plasma Applications inAir-Sterilization,” International Symposium on Plasma Science (August2005), a non-thermal plasma emission device and method for treatingairborne biologic contaminants was proposed, but neither tested norsufficiently described such that one of ordinary skill in the art wouldbe able to reproduce the proposed technology and in flight plasmasterilization methodology. The publication discloses a calibration testusing a Pathogen Detection and Remediation Facility (PDRF) incorporatinga plasma emission device such as a Dielectric Barrier Discharge (DBD)device or a Magnetically-Rotated Gliding Arc (MRGA) device. Thecalibration experiment involved emission of cyanobacterial aerosol toidentify bioaerosol losses from diffusion, inertia and evaporation toestablish accurate controls before applying non-thermal plasma.Additionally, the publication proposes, but does not describe,sterilization experiments with cyanobacteria and influenza A virus. Thepublication does not apply plasma to the cyanobacteria calibrationexperiments.

Based on the known efficiency of plasma-based sterilization technology,it is unexpected that it would be possible to render a substantialproportion of biologic agents inactive in flight in a short time, suchas milliseconds, using non-thermal plasma. By comparison, DBD surfacesterilization treatment times are often 1000 times longer, on the orderof seconds, and in some cases even minutes in duration.

Therefore it would be desirable to develop a method capable ofefficiently sterilizing airborne biologic agents within a period ofmilliseconds.

SUMMARY OF THE INVENTION

The invention is directed to a method for sterilizing biologic agents ina gaseous media. In one aspect, the invention is a method for treatingculturable E. coli in a moving air stream at high airflow rates withplasma so as to achieve a substantial reduction in active bacteriawithin a period of milliseconds.

In another aspect, the invention is directed to a method for effectivelysterilizing concentrated bacterial bioaerosols in a moving air stream athigh airflow rates with plasma.

In another aspect, the invention cooperates with a Heating, Ventilationand Air Conditioning (HVAC) system for one or more of the purposes ofpreventing bioterrorism, preventing the spread of contagious or toxicagents, containing airborne pandemics, sterilizing spaces and buildings,treating exhaust ventilation streams, minimizing environmentalpollutants, improving general air quality and preventing sick buildingsyndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the Pathogen Detection and Remediation Facility(PDRF).

FIG. 2 is a depiction of a DBD gaseous media sterilization device.

FIG. 3 is a graph of a current and voltage waveform of the DBD device.Lower curve is voltage signal in kilovolts (kV), upper curve is currentsignal in amperes (A).

FIG. 4 is a graph of colony forming units versus time showing theresults of DBD-treated E. Coli in a PDRF system in three replicatetrials.

FIG. 5 is a flow-cytometric histogram for the total number of E. Coli(alive+dead) stained by SYBR Green I.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Non-thermal plasma is an electrically neutral mixture of atoms,molecules, electrons and ions that cannot be described by onetemperature. Average energy of electrons in non-thermal plasma isusually on the level of more than 1 electron-volt (cV), (1 eVcorresponds to temperature of about 11,600 K), while averagetranslational temperature of heavy particles (ions, molecules and atoms)is much less, usually below 3,000 K and often is very close to ambient(room) temperature, e.g. 20° C.

For the purpose of this invention it is possible to consider abiological agent as an active one if it is capable of reproduction orproliferation (so-called culturable microorganisms) in a specialappropriate media or in human organism. If microorganism is not able toreproduce itself (is not culturable), it is highly probable that itcannot harm another organism even if its structure is mechanicallyintact, and thus such microorganisms are considered to be inactivated.

This invention is directed to a method for sterilizing biologiccontaminants entrained, dispersed or suspended in a gaseous media athigh flow rates by plasma emissions and a system for carrying out themethod.

One system of the invention may include a Pathogen Detection andRemediation Facility (PDRF) and a plasma emission device, which rendersbiologic contaminants non-culturable. The PDRF, as shown in FIG. 1, is aspecialized plug flow reactor that is capable of circulating andsampling a gaseous media as well as bioaerosol generation, capture andcontainment. It is designed to operate at high airflow rates of about 25L/s or greater, which are typical for indoor ventilation systems. Acentrifugal blower 2 drives and circulates the contaminated gaseousmedia through a mixing chamber 3 and ventilation line 1. In a preferredembodiment, the PDRF is a closed system that allows for humiditycontrol, which may be set to optimize sterilization capabilities. Theair pressure and temperature within the line is regulated by a pressurerelease value 4 and hygrometer/thermometer 5. One of the primaryadvantages of the PDRF recirculating airflow system the ability torepeatedly treat bioaerosols, entrained, dispersed or suspended in agaseous media, recirculating through the plasma emission device 6. Thesystem also includes a bioaerosol nebulizer 7 and compressed air source8 for the purposes of introducing a sample of biologic contaminantsentrained, dispersed or suspended in a gaseous media.

The plasma emission device 6 may include any mechanism capable ofproducing plasma in a directed air or aerosol stream. In a preferredembodiment, the device may be a Dielectric Barrier Discharge ventilationgrating (DBDG) plasma device or a Magnetically-Rotated Gliding Arc(MRGA) device. FIG. 2 shows a DBDG device 6, which includes a pair ofcomb-like electrodes inserted one into another 16 DBDG consists of twocomb-like gratings (grills) inserted one into another. 1-mm wires 13 ofone grating are covered with quartz capillaries of 2 mm outer diameterand connected to a high voltage AC power supply 15. There are 1.5 mm airgaps between these insulated wires and 1-mm bare wires 13 of the secondgrating that are grounded. When high-voltage AC power supply is on,non-equilibrium plasma is generated in the air gaps between bare andinsulated wires, connected to a high voltage source 15, air samplingports 9, connected to a set of liquid impingers 12 and a vacuum source14.

In a preferred embodiment, a DBD device 6 is use to generate plasma. TheDBD is an alternating current discharge between two electrodes 16, atleast one of which is covered by a dielectric. DBD plasma can be formedin the gas filled area, otherwise known as the discharge gap, betweenone electrode and a dielectric or between two dielectrics. The DBD isdriven by an applied alternating high voltage (several kilovolts), whichgenerates a high electric field between the electrodes 16. In theabsence of a dielectric, the discharge starting from the first spark,would rapidly progress to an arc, as the electrons in the spark wouldinitiate a series of ionization events, leading to very high current andultimately to arc formation. The dielectric prevents arc formation byaccumulating charge on the surface and generating an electric field thatopposes the applied field, thereby limiting the current and preventingdevelopment of an uncontrolled discharge. Alternating high voltagepolarities ensures formation of this discharge in each half of thevoltage cycle.

Usual DBD devices 6 operate in the kilohertz range, so plasma betweenthe electrodes 16 does not have enough time to extinguish completely. Inone embodiment, the non-thermal plasma discharge is generated by a highfrequency oscillation of high voltage that is from about 1 to about20,000 kHz, optionally, from about 5 to about 30 kHz, and a peak-to peakvoltage of about 1 to about 50 kV, optionally, from about 5 to about 30kV.

As a gaseous media in which biologic contaminants are entrained,dispersed or suspended is introduced into the DBD device 6 through anentry port 10, a quasi-sinusoidal waveform, as shown in FIG. 3, isgenerated by a quasi-pulsed high-voltage source and applied across theelectrode gaps generating a high electric field and non-equilibriumplasma that covers the whole area between electrodes 16. The periodbetween pulses is approximately 600 μs, peak-to-peak voltage is 28 kV,and current reaches nearly 50 amps in a pulse. The average power of thedischarge is approximately 330 watts and considering the discharge areaof 91 cm², the power density is 3.6 watts/cm². The majority of power isdischarged in the very short duration of the pulse itself, which has aperiod of 77 μs and average pulse power of 2618 W. Since the residencetime of a bioaerosol particle passing through the discharge area is 0.73ms and the period between pulses is 0.6 ms, this means that eachbioaerosol particle that passes through the DBD discharge experiencesabout 1 pulse of DBD discharge power, assuming the discharge is fairlyuniform and gaps between streamers are not considered. The air passesthrough the plasma stream and leaves the DBD through an exit port 11. Ina preferred embodiment, the plasma “curtain” that is created should nothave large holes, e.g. larger than distance between DBD surfaces. Thetime between high voltage pulses that generate plasma should not besignificantly larger then the residence time of air in plasma t=Sd/Q,where S is the free area where plasma is generated, d is the thicknessof the bare electrodes and Q is the air flow rate. The power should besufficient to provide the desired degree of sterilization.

The higher the power, the greater the degree of sterilization. Forexample, an instantaneous decrease in E. Coli concentration of a factorof 30 is observed at an energy input of about 330 W/25 l/s=13.2 J/l(joule per liter). Thus, about 27 J/l should provide a 3 log reductionin E. Coli concentration, 9 J/l should provide a 1 log reduction and 54J/l should provide a 6 log reduction in plasma. Additional sterilizationmay also occur in the post-plasma treatment flow. Accordingly, preferredranges for operation are about 3-100 J/l and, more preferably, 5-30 J/l.

The method of the present invention involves sterilizing biologiccontaminants entrained, dispersed or suspended in a gaseous media bycirculating contaminated gaseous media through a stream of plasma.Microorganisms are rendered inactive within milliseconds and thecontaminants do not have to adhere or contact a surface to enablesterilization. Flow rates can be increased by increasing input power.The residence time should be sufficient to ensure that at least onecurrent pulse occurs during the residence time to thereby ensure plasmatreatment of all flow. More preferably, the time between plasmageneration pulses should be less than the residence time, and morepreferably, less than half the residence time. In comparison tocurrently known methods for air sterilization, the present inventionenables sterilization of biologic agents in a gaseous flowing mediawithout the use of filters or other sterilization surfaces. This isunexpected since plasma sterilization in a gaseous flowing media issignificantly more difficult and challenging than sterilization insolution or on a surface because of the very short residence time ofmicroorganisms in the plasma zone. The methods of the prior art aretherefore unable to explain the single run, 2-log reduction in viable E.Coli population. Additionally, based on the prior art and theoreticalmodels, one of ordinary skill in the art would expect efficiency returnsof in flight plasma based sterilization to be no more than about 15%after one application of plasma emission for the experiment describedabove. The 97% efficiency of bacteria inactivation shown in FIG. 4, istherefore quite surprising. Moreover, the invention only requires aminimal amount of energy for operation, resulting in low operationalcosts.

Experimental experience shows that is very difficult to createbio-contamination in large volume of air with concentration higher than1,000,000 particles per 1 liter. Thus, a 5 or greater log reductionshould be sufficient to prevent consequences of a biological attack. Inother cases (pandemic, sick building syndrome) disinfection maytypically be accomplished by a 3 or greater log reduction.

It is envisioned that this technology will be used to improve airventilation and sterilization by deactivating airborne microorganisms.The technology is particularly well suited for applications in heating,ventilation and air conditioning (HVAC) systems. More specifically, theinvention may be used in the bioterrorism defense industry to preventthe spread of toxic or infectious agents, contain airborne pandemicssuch as avian flu, sterilize various spaces such as hospitals,pharmaceutical plants and manufacturing facilities, treat exhaustventilation streams, minimize environmental pollutants in industrialsettings, improve general air quality, and prevent sick buildingsyndrome.

Examples Example 1

The PDRF system, shown in FIG. 1, is a bioaerosol treatment facilitydesigned to provide a recirculating gaseous media environment. Becausethe PDRF system is capable of recirculating airflow, bioaerosolsentrained, dispersed or suspended in the gaseous media can be treatedwith repeated passes through the same plasma discharge. Additionally, asealed, recirculating system allows for complete control over relativehumidity inside the system, which is important because even smallfluctuations in relative humidity have been shown to significantlydecrease the survivability of airborne bacteria. The PDRF system wasdesigned as a plug flow reactor, such that airflow inside the system isturbulent, minimizing radial variation of the bacterial concentration inthe airflow.

The PDRF system has a total volume of 250 liters and is designed tooperate at high airflow rates of at least 25 L/s, which is typical ofindoor ventilation systems. The system has an inlet with an attachedCollison nebulizer for bioaerosol generation and two air-sampling portsconnected to a vacuum air sampling system. The system also has a largemixing chamber that contains a series of aluminum baffle plates and avariable speed centrifugal blower motor 2 that drives the air throughthe DBD treatment chamber 6. The residence time, that is, the time forone bioaerosol particle to make one complete revolution through thesystem, is approximately 10 seconds.

The DBD device 6, shown in FIG. 2, may include of a thin plane of wireswith equally spaced air gaps of 1.5 mm, and each second wire is a highvoltage electrode 16. The high voltage electrodes 16 are about 1 mmdiameter copper wires shielded with a quartz capillary dielectric thathas an approximate wall thickness of 0.5 mm. The total area of the DBDincluding electrodes is 214.5 cm² and without electrodes is 91.5 cm².The DBD device 6 further has two air sample ports 9 located at adistance of 10 cm from each side of the discharge area so thatbioaerosol can be sampled immediately before and after it enters theplasma discharge. When the PDRF system is operated at a flow rate of 25L/s, the air velocity inside the DBD chamber is 2.74 m/s and theresidence time of one bioaerosol particle, containing one E. Colibacterium, passing through the DBD is approximately 0.73 milliseconds.

The DBD device 6 is operated using a quasi-pulsed power supply 15 thatdelivers a quasi-sinusoidal voltage waveform with a very fast rise timethat nearly simulates a true square wave pulse, as shown in FIG. 3. Theperiod between pulses is approximately 600 μs, peak-to-peak voltage is28 kV, and current pulses that pass through air plasma reach 50 amps.The average power of the discharge is approximately 330 watts. Theaverage discharge area is approximately 91 cm², and the average powerdensity is 3.6 watts/cm². The majority of power is discharged in thevery short duration of the pulse itself, which has a period of 77 μs andaverage pulse power of 2618 W. Since the residence time of a bioaerosolparticle passing through the discharge area is 0.73 ms and the periodbetween pulses is 0.6 ms, assuming the discharge is fairly uniform andthere are no gaps between streamers, each bioaerosol particle thatpasses through the DBD experiences about 1 pulse of DBD power.

Example 2

The effectiveness of the present method was tested on Escherichia Coli(K-12 strain). The present invention was found to effectively render 99%of the viable E. Coli inactive.

Each culture of E. Coli (K-12 strain) used in this study was prepared ina 250 ml flask containing LB growth medium and was incubated overnightfor about 12 hours at 37° C. with constant mixing on a water bathshaker. The culture was then centrifuged at 3500 rpm for a period of 10minutes and rinsed with a sterile phosphate buffered saline (PBS)solution (1× concentration). This procedure was repeated three times toensure complete removal of growth medium and to concentrate the cultureto a volume of 45 ml for nebulization.

After the PDRF system was pre-sterilized using the internal heatingsystem 5 and pre-humidified to a relative humidity of about 70%, thebacteria culture was placed into a BGI 24-jet Collison nebulizer, andthe nebulizer was operated at 40 psi for a period of 45 seconds at anebulization rate of about 1.1 ml/min. The DBD device 6 was thenswitched on for a period of 10 seconds, and the first two air sampleswere immediately and sequentially taken before and after plasmatreatment. The discharge time of 10 seconds was selected to correspondwith the residence time of the bioaerosol to make one completerevolution in the PDRF system. The samples measure the decontaminationeffectiveness of DBD on a per pass basis, ensuring that each bioaerosolparticle has been treated once by the discharge. Another set of airsamples was taken approximately 2 minutes later to accommodate theamount of time required to remove and replace the air samplers with thenext set of pre-sterilized samplers. This process was repeated againuntil the typical number of air samples, approximately six, wasachieved. Each of the pre-sterilized air samplers was initially filledwith 30 ml of sterile PBS solution, and after sampling, each solutionwas transferred to a sterile 50 ml centrifuge tube for assaying.

Following each experiment, liquid samples from each impinger 12 weretransferred to 50 ml centrifuge tubes and serially diluted in PBS,plated onto LB agar plates, and incubated at 37° C. overnight. Visiblecolonies were counted and recorded within the following 24-hour period.

Additionally, a fluorescent method of bacteria detection, flowcytometry, was also employed to detect the presence of E. Coli in eachair sample taken during experiments. It is important to verify thepresence of inactivated bacteria because it eliminates the possibilitythat DBD could act as an electrostatic precipitator, which could chargethe bioaerosol droplets and remove them from the airflow inside the PDRFsystem. Whereas colony counting techniques are limited to detecting onlyviable bacteria, flow cytometry is capable of detecting the presence ofbacteria whether it is viable or inactivated. This technique utilizestwo fluorescent dyes; SYBR Green I to detect the presence of allbacteria (dead and alive), and propidium iodide (PI) to detect thebacteria with disintegrated cyto-plasmic membranes.

FIG. 5 shows flow cytometry results for the DBDG-treated air samplesusing only SYBR Green I florescent dye. The florescent intensity peakfor air samples one through six is identical, which means that there arethe same number of total bacteria present for each air sample takenduring experiments. The stock positive control sample is a pureunaltered sample of E. Coli whose intensity peak was two orders of valuegreater than the intensity of the air samples. Additionally, theintensity of propidium iodide red fluorescence (not shown) was found tobe negligible in comparison with expected PI positive control andtherefore the outer membranes of E. Coli were not disintegrated afterinteraction with DBDG.

The cytometry results show that DBD plasma is acting not as anelectrostatic precipitator, but as a device capable of decontaminatinghigh concentrations of bacterial bioaerosol in flight at high flow ratesin a laboratory scale ventilation system.

An interesting yet unexpected decrease in bacterial concentration isshown between samples 2 and 3 of the DBD-treated trial in which thenumber of viable bacteria dropped to zero. It is not immediately clearhow such a drastic change could have occurred especially since theplasma discharge was turned off during this period. Additionally, it isnot fair to claim that this second drop in concentration is anadditional 4-log reduction because the sampling system efficiency is low(6%±3%) and zero bacteria present in the air samplers could mean thatthere are indeed viable bacteria present in the system, but its out ofthe range of our detection limits.

Having described the preferred embodiments of the invention which areintended to be illustrative and not limiting, it is noted thatmodifications and variations can be made by persons skilled in the artin light of the above teachings. It is therefore to be understood thatchanges may be made in the particular embodiments of the inventiondisclosed which are within the scope and spirit of the invention asoutlined by the appended claims. Having thus described the inventionwith the details and particularity required by the patent laws, theintended scope of protection is set forth in the appended claims.

1. A method for sanitizing biologic contaminants entrained, dispersed orsuspended in gaseous media comprising the step of: contacting a biologicagent with a non-thermal plasma discharge entrained, dispersed orsuspended in gaseous media for a sufficient time to render a substantialportion of said biologic agent inactive.
 2. The method of claim 1,wherein the biologic agent is contacted with said plasma.
 3. The methodof claim 1, wherein the non-thermal plasma is generated in a dielectricbarrier discharge.
 4. The method of claim 1, wherein the non-thermaldielectric barrier discharge plasma discharge is generated by a highfrequency oscillation of about 1 kHz to about 20,000 kHz.
 5. The methodof claim 1, wherein the non-thermal plasma discharge is generated by ahigh frequency oscillation of about 5 kHz to about 30 kHz.
 6. The methodof claim 4, wherein the high frequency oscillation is generated byapplying a voltage of about 1 kV to about 50 kV.
 7. The method of claim4, wherein the high frequency oscillation is generated by applying avoltage of about 5 kV to about 30 kV.
 8. The method of claim 1 whereinthe biologic agent is selected from the group consisting of at least onebacterial agent, at least one viral agent, at least one spore agent anda mixture thereof.
 9. A system including a device for directing a flowof gaseous media, and a plasma generation device for generation of anon-thermal plasma in contact with a gaseous media flowing in saidflow-directing device, whereby a substantial proportion of a biologicagent in said gaseous media is inactivated by contact with saidnon-thermal plasma.
 10. The system of claim 9, wherein said system is aheating, ventilation or air conditioning system.
 11. The system of claim9, wherein said plasma generation device comprises a dielectric barrierdischarge grating device.
 12. The system of claim 9, wherein thenon-thermal plasma generation device is capable of generating a highfrequency plasma of about 1 kHz to about 20,000 kHz.
 13. The system ofclaim 9, wherein the non-thermal plasma discharge is generated by a highfrequency oscillation of about 5 kHz to about 30 kHz.
 14. The system ofclaim 9, wherein the non-thermal plasma generation device does notinclude a filter.